Bioinks play a central role in 3D-bioprinting by providing the supporting environment within which encapsulated cells can endure the stresses encountered during the digitally driven fabrication process and continue to mature, proliferate, and eventually form extracellular matrix (ECM). In order to be most effective, it is important that bioprinted constructs recapitulate the native tissue milieu as closely as possible. As such, musculoskeletal soft tissue constructs can benefit from bioinks that mimic their nanofibrous matrix constitution, which is also critical to their function. This study focuses on the development and proof-of-concept assessment of a fibrous bioink composed of alginate hydrogel, polylactic acid nanofibers, and human adipose-derived stem cells (hASC) for bioprinting such tissue constructs. First, hASC proliferation and viability were assessed in 3D-bioplotted strands over 16 days in vitro. Then, a human medial knee meniscus digitally modeled using magnetic resonance images was bioprinted and evaluated over 8 weeks in vitro. Results show that the nanofiber-reinforced bioink allowed higher levels of cell proliferation within bioprinted strands, with a peak at day 7, while still maintaining a vast majority of viable cells at day 16. The cell metabolic activity on day 7 was 28.5% higher in this bioink compared to the bioink without nanofibers. Histology of the bioprinted meniscus at both 4 and 8 weeks showed 54% and 147% higher cell density, respectively, in external versus internal regions of the construct. The presence of collagen and proteoglycans was also noted in areas surrounding the hASC, indicating ECM secretion and chondrogenic differentiation.

A critical consideration in tissue engineering is to recapitulate the microstructural organization of native tissues that is essential to their function. Scaffold-based techniques have focused on achieving this via the contact guidance principle wherein topographical cues offered by scaffold fibers direct migration and orientation of cells to govern subsequent cell-secreted extracellular matrix organization. Alternatively, approaches based on acoustophoretic, electrophoretic, photophoretic, magnetophoretic, and chemotactic principles are being investigated in the biofabrication domain to direct patterning of cells within bioink constructs. This work describes a new acoustophoretic three-dimensional (3D) biofabrication approach that utilizes radiation forces generated by superimposing ultrasonic bulk acoustic waves (BAW) to preferentially organize cellular arrays within single and multi-layered hydrogel constructs. Using multiphysics modeling and experimental design, we have characterized the effects of process parameters including ultrasound frequency (0.71, 1, 1.5, 2 MHz), signal voltage amplitude (100, 200 mVpp), bioink viscosity (5, 70 cP), and actuation duration (10, 20 min) on the alignment characteristics, viability and metabolic activity of human adipose-derived stem cells (hASC) suspended in alginate. Results show that the spacing between adjacent cellular arrays decreased with increasing frequency (p < 0.001), while the width of the arrays decreased with increasing frequency and amplitude (p < 0.05), and upon lowering the bioink viscosity (p < 0.01) or increasing actuation duration (p < 0.01). Corresponding to the computational results wherein estimated acoustic radiation forces demonstrated a linear relationship with amplitude and a nonlinear relationship with frequency, the interaction of moderate frequencies at high amplitudes resulted in viscous perturbations, ultimately affecting the hASC viability (p < 0.01). For each combination of frequency and amplitude at the extremities of the tested range, the hASC metabolic activity did not change over 4 d, but the activity of the low frequency-high amplitude treatment was lower than that of the high frequency-low amplitude treatment at day 4 (p < 0.01). In addition to this process-structure characterization, we have also demonstrated the 3D bioprinting of a multi-layered medial knee meniscus construct featuring physiologically-relevant circumferential organization of viable hASC. This work contributes to the advancement of scalable biomimetic tissue manufacturing science and technology.

3D bioprinting has recently emerged as a very useful tool in tissue engineering and regenerative medicine. However, developing suitable bioinks to fabricate specific tissue constructs remains a challenging task. Herein, we report on a nanocellulose/chitosan-based bioink, which is compatible with a 3D extrusion-based bioprinting technology, to design and engineer constructs for bone tissue engineering and regeneration applications. Bioinks were prepared using thermogelling chitosan, glycerophosphate, hydroxyethyl cellulose, and cellulose nanocrystals (CNCs). Formulations were optimized by varying the concentrations of glycerophosphate (80−300 mM), hydroxyethyl cellulose (0−0.5 mg/mL), and CNCs (0−2% w/v) to promote fast gelation kinetics (<7 s) at 37 °C and retain the shape integrity of constructs post 3D bioprinting. We investigated the effect of CNCs and pre-osteoblast cells (MC3T3-E1) on the rheological properties of bioinks, bioink printability, and mechanical properties of bioprinted scaffolds. We demonstrate that the addition of CNCs and cells (5 million cells/mL) significantly improved the viscosity of bioinks and the mechanical properties of chitosan scaffolds post-fabrication. The bioinks were biocompatible and printable at an optimized range of printing pressures (12−20 kPa) that did not compromise cell viability. The presence of CNCs promoted greater osteogenesis of MC3T3-E1 cells in chitosan scaffolds as shown by the upregulation of alkaline phosphatase activity, higher calcium mineralization, and extracellular matrix formation. The versatility of this CNCs-incorporated chitosan hydrogel makes it attractive as a bioink for 3D bioprinting to engineer scaffolds for bone tissue engineering and other therapeutic applications.

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